57Fe and 1H electron-nuclear double resonance of three doubly reduced states Escherichia coli sulfite reductase

We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the 57Fe hyperfine interactions in the bridged-siroheme [4Fe-4S] cluster that forms the catalytically active center of the two-electron-reduced hemoprotein subunit of Escherichia coli NADPH-sulfite reductase (SiR2-). Previous electron paramagnetic resonance (EPR) and M?ssbauer studies have shown that this enzyme oxidation state can exist in three distinct spectroscopic forms: (1) a "g = 2.29" EPR species that predominates in unligated SiR2-, in which the siroheme Fe2+ is believed to be in an S = 1 state; (2) a "g = 4.88" type of EPR species that predominates in SiR2- in the presence of small amounts of guanidinium sulfate, in which the siroheme Fe2+ is in an S = 2 state; and (3) a classical "g = 1.94" type of EPR species that is seen in SiR2- ligated with CO, in which the siroheme Fe2+ is in an S = 0 state. In all three species, the cluster is in the [4Fe-4S]1+ state, and two distinct types of Fe site are seen in M?ssbauer spectroscopy. ENDOR studies confirm the M?ssbauer assignments for the cluster 57Fe in the g = 1.94 state, with A values of 37, 37, and 32 MHz for site I and ca. 19 MHz for site II. The hyperfine interactions are not too different on the g = 2.29 state, with site I Fe showing more anisotropic A values of 32, 24, and 20 MHz (site II was not detected).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of temperature and photoperiod upon adult eclosion of the sweetpotato whitefly, Bemisia tabaci

A series of experiments were conducted to characterize patterns of eclosion by Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) to their adult stage and to determine how these patterns are influenced by certain environmental parameters. Under a constant temperature of 29.5±0.6°C and a photoperiod of 14:10LD, 90% of the adults emerged from their pupal cases between 0600 and 0930 h (with lights on occurring at 0600 h). Few emerged during hours of darkness. The peak time of adult emergence was delayed when temperatures were fluctuated. Under a series of constant temperatures, a significant inverse correlation was found between the time of median emergence (i.e., eclosion of 50% of the total number of adults) and temperature (P<0.001). No emergence was observed at temperatures below 17±0.3°C. Emergence patterns persisted under conditions of continuous light and continuous darkness, suggesting the presence of a circadian system.

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Zusammenfassung Um das Verständnis über den Lebenslauf von Bemisia tabaci zu ergänzen, wurde eine Serie von Experimenten durchgeführt, deren Zweck die Charakterisierung des Ausschlüpfvorgangs in das Endstadium war und die Feststellung, wie dieser Vorgang von gewissen Umweltparametern beeinflusst wird. Bei einer konstanten Temperatur von 29.5±0.6°C und einem Beleuchtungszyklus von 14: 10 LD (Licht/Dunkelheit) schlüpften 90% der Ausgewachsenen zwischen 0600 Uhr and 0930 Uhr (ab 0600 Uhr mit Licht) aus ihren Puppenhüllen aus. Wenig Ausschlüpfen geschah während der unbeleuchteten Stunden. Der Höhepunkt des Ausschlüpfens wurde bei wechselnden Temperaturen verschoben. Bei einer Serie von gleichbleibenden Temperaturen wurde eine bedeutende inverse Korrelation zwischen der medianen Ausschlüpfzeit (d.h. 50% der gesamten Ausgewachsenen schlüpften aus) und der Temperatur festgestellt (P<0.001). Kein Ausschlüpfen wurde beobachtet bei Temperaturen unter 17°C. Das Ausschlüpfschema war gleichbleibend bei dauerndem Licht oder dauernder Dunkelheit, was auf das Vorhandensein eines circadianen Systems hinweist.

     

Reversal of Con A-induced suppression by a platelet-derived factor which binds to activated suppressor T cells

A platelet-derived factor found in serum as well as in platelet releasate prepared either with calcium ionophore or with thrombin was shown to reverse Con A-induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) in vivo in (CB6)F1 mice. In addition, as shown previously, lymphoma cell-induced suppression in SJL mice was similarly reversed. The factor could be injected prior to Con A on the day before SRBC injection, or on the same day as antigen with comparable results. It also enhanced PFC responses in the absence of Con A. Suppressor cell induction by Con A in vivo, as demonstrated by assay on PFC responses of normal spleen cells in vitro, was abrogated by simultaneous injection of the platelet factor. Cells from mouse spleen and lymph node, but not from thymus could absorb the factor from human serum at 4 degrees C. The phenotype of the relevant spleen cells was L3T4-, Ly1-, Ly2+, Thy1+, Ly22+, Qa1+, Qa4+, Qa5+, and Ly6.IE+. These results suggest that this factor binds to activated peripheral T cells of the suppressor cell phenotype.     

Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from approximately 0.2 to 50 were set and maintained (for up to 45 min) in this system. ATP and ADP concentrations were controlled by the addition of either a phosphoarginine- or phosphocreatine-based regenerating system. Ouabain-sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition. Na/K exchange was found to increase to saturation with ATP (K 1/2 approximately equal to 250 microM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K 1/2 approximately equal to 350 microM). The elevation of ATP from approximately 100 to 1,800 microM did not appreciably affect Na/Na exchange. In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange. The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na. Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate-limited by the conformational change associated with the release of occluded K [E2 X (K) X ATP----E1 X ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form. A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (E1). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E1P X (Na). Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K pump operates.     

Antibody-induced antigenic modulation is antigen dependent: characterization of 22 proteins on a malignant human B cell line

Expression of several of the surface antigens on normal and malignant hematopoietic cells is reduced or is modulated by incubation with specific antibodies. Although antigenic modulation provides a means by which cells can escape antibody-mediated immune destruction, the physiologic significance and frequency of this phenomenon are both poorly understood. To begin to address these issues, we identified and characterized surface antigens on the malignant B cell line Laz 221 established from a patient with acute lymphoblastic leukemia (ALL). Indirect immunofluorescence analysis with the use of 26 hematopoietic cell populations and immune precipitation studies with the use of iodinated ALL cells indicate that 163 monoclonal antibodies (MoAb) identify 22 different proteins on this cell line, including at least six previously described surface molecules. Seven of these antigens are expressed by all nucleated cells examined, whereas only the mu chain of immunoglobulin is B cell specific. Incubation of specific MoAb with cultures of Laz 221 cells at 37 degrees C reduces or modulates surface expression of five of these 22 antigens (p45, immunoglobulin mu chain, transferrin receptor, common ALL antigen (CD10), and p105). Studies that made use of multiple MoAb specific for the same antigen suggest that the capacity for antigenic modulation is an intrinsic property of individual antigens. These studies also suggest that the murine immune response to shared human antigens varies from one immunizing cell population to another. For example, three of the antigens present on Laz 221 cells were only identified by MoAb raised to the Burkitt's cell line Ramos and vice-versa. Only one of these six shared antigens is present in greater amounts on the immunogenic cell population. Immunogenicity of individual human antigens in the mouse may be a function of their cell surface environment.     

Characterization of a progressive neurodegenerative disease induced by a temperature-sensitive Moloney murine leukemia virus infection.

A progressive neurodegenerative disease occurred following infection of mice with a temperature-sensitive (ts) isolate of Moloney (Mo) murine leukemia virus (MuLV), ts Mo BA-1 MuLV. This NB-tropic ecotropic MuLV, which was ts for a late function, induced a syndrome of tremor, weakness of the hind limbs, and spasticity following infection of several strains of laboratory neonatal mice, including NFS, C3H/He, CBA, SJL, and BALB/c. The latent period of 8 to 16 weeks was considerably longer than that observed for the acute paralytic diseases observed following neonatal infection with other ts Mo-MuLV, rat-passaged Friend MuLV, and some wild mouse ecotropic MuLVs. Spongiform pathology without inflammation and degeneration of neurons devoid of budding virions occurred in the cerebellar grey matter, brain stem, and upper spinal cord; but lower spinal cord anterior horn cells were less obviously affected than in other MuLV-associated neuroparalytic syndromes. ts Mo BA-1 MuLV differed from other ts Mo-MuLV mutants that are capable of inducing a neuroparalytic syndrome in that while infected nervous system tissue contained high levels of MuLV p30 and gp70, no evidence of precursor accumulation or abnormal processing of MuLV p30 or gp70 could be demonstrated. The localization of virus within the nervous system suggests that direct neuronal infection may not be the etiologic mechanism in this MuLV-induced neurodegenerative disease.     

Differential contributions of Ng-CAM and N-CAM to cell adhesion in different neural regions

Individual neurons can express both the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) at their cell surfaces. To determine how the functions of the two molecules may be differentially controlled, we have used specific antibodies to each cell adhesion molecule (CAM) to perturb its function, first in brain membrane vesicle aggregation and then in tissue culture assays testing the fasciculation of neurite outgrowths from cultured dorsal root ganglia, the migration of granule cells in cerebellar explants, and the formation of histological layers in the developing retina. Our strategy was initially to delineate further the binding mechanisms for each CAM. Antibodies to Ng-CAM and N-CAM each inhibited brain membrane vesicle aggregation but the binding mechanisms of the two CAMs differed. As expected from the known homophilic binding mechanism of N-CAM, anti-N- CAM-coated vesicles did not co-aggregate with uncoated vesicles. Anti- Ng-CAM-coated vesicles readily co-aggregated with uncoated vesicles in accord with a postulated heterophilic binding mechanism. It was also shown that N-CAM was not a ligand for Ng-CAM. In contrast to assays with brain membrane vesicles, cellular systems can reveal functional differences for each CAM reflecting its relative amount (prevalence modulation) and location (polarity modulation). Consistent with this, each of the three cellular processes examined in vitro was preferentially inhibited only by anti-N-CAM or by anti-Ng-CAM antibodies. Both neurite fasciculation and the migration of cerebellar granule cells were preferentially inhibited by anti-Ng-CAM antibodies. Anti-N-CAM antibodies inhibited the formation of histological layers in the retina. The data on perturbation by antibodies were correlated with the relative levels of expression of Ng-CAM and N-CAM in each of these different neural regions. Quantitative immunoblotting experiments indicated that the relative Ng-CAM/N-CAM ratios in comparable extracts of brain, dorsal root ganglia, and retina were respectively 0.32, 0.81, and 0.04. During culture of dorsal root ganglia in the presence of nerve growth factor, the Ng-CAM/N-CAM ratio rose to 4.95 in neurite outgrowths and 1.99 in the ganglion proper, reflecting both polarity and prevalence modulation. These results suggest that the relative ability of anti-Ng-CAM and anti-N-CAM antibodies to inhibit cell-cell interactions in different neural tissues is strongly correlated with the local Ng-CAM/N-CAM ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

Site-restricted expression of cytotactin during development of the chicken embryo

The sequential appearance of the extracellular matrix (ECM) protein, cytotactin, was examined during development of the chicken embryo by immunohistochemical techniques. Although cytotactin was identified as a molecule that mediates glia-neuron interactions, preliminary immunohistochemical localization of the molecule suggested that it was an ECM protein with a widespread but nonetheless more restricted distribution than either fibronectin or laminin. In the present study, it was found that cytotactin is first present in the gastrulating chicken embryo. It appears later in the basement membrane of the developing neural tube and notochord in a temporal sequence beginning in the cephalic regions and proceeding caudally. Between 2 and 3 d of development, the molecule is present at high levels in the early neural crest pathways (surrounding the neural tube and somites) but, in contrast to fibronectin and laminin, is not found in the lateral plate mesoderm or ectoderm. At later times, cytotactin is expressed extensively in the central nervous system, in lesser amounts in the peripheral nervous system, and in a number of nonneural sites, most prominently in all smooth muscles and in basement membranes of lung and kidney. Cytotactin appears in adult tissues with distributions that are similar to those seen in embryonic tissues. The findings raise the possibility that certain ECM proteins contribute to pattern formation in embryogenesis as a result of their restricted expression in a spatiotemporally regulated fashion at some sites but not at others.     

Interaction of radiation-generated radicals with myoglobin in aqueous solution. III. Effects of oxygen and catalase on the product distribution in solutions of ferrimyoglobin containing N2O

The gamma-radiolysis of aqueous solutions of ferrimyoglobin in the presence of N2O at pH 7.3 has been examined as a function of added catalase and oxygen. Changes in the nature of the heme group have been monitored by visible absorption spectrophotometry and analysed quantitatively by a multiple wavelength method based on Beer's Law. Simple chemical analyses have been used to confirm qualitative identification of the product derivatives. As observed previously, the ferriheme is reduced by indirect globin-mediated action initiated by OH/H. The yield of reduced product decreases as [O2] increases. Conversion to ferrimyoglobin through the participation of H2O2 derived from irradiated water and from protein-mediated processes in oxygenated solution, is eliminated by the presence of catalase. Formation of a hemichrome form of ferrimyoglobin is apparent at higher doses in the presence of O2. These results demonstrate that oxygen plays an important role in controlling the nature and extent of redox that manifests ultimately on the heme group of ferrimyoglobin as a result of the initial interaction of OH/H.